Foodborne diseases have attracted great attentions as food safety is one of the major concerns to public health. Therefore, quick and sensitive detection equipments are extremely essential for food hygiene inspection. Similarly, for food processing enterprises and food supervision departments, quick detection of the quantity of target bacteria in the food is favorable for improvement of monitoring of food quality and effective public food hygiene and safety protection.
Presently, conventional methods for detection of foodborne pathogens include biochemical identification, Enzyme-Linked Immunosorbent Assay (ELISA), PCR and so on. However, such methods have certain disadvantages, such as complicated preliminary processing of samples and extraction of DNA, long detection cycle, low detection sensibility and the lack of economical, convenient, quick and sensitive detection techniques. On the contrary, biosensor technology based on micro-fluidic chip has become a R&D focus owing to such advantages as high flux, miniaturization, automation and easy integration.
As micro-fluidic chip can integrate complicated chemical processes into a chip, numerous quick detection equipment based on micro-fluidic chip have appeared in recent years, such as electrochemical micro-fluidic chip, impedance micro-fluidic chip and so on. According to their detection principles, most of such chips aim to detect the quantity of bacteria through measurement of variation to the current or resistance of micro-fluidic equipments before and after the detection of target bacteria. The advantage of such equipment lies in the fact that it can analyze extremely low quantity of bacteria. However, such equipment is not easy for operation and is relatively expensive.
Another type of equipment taking the micro-fluid as the basis for detection is based on optic detection principles (fluorescent staining or color variation). The advantage of such detection mechanism lies in the fact that detection signal can be directly observed and detected to realize quantification of target bacteria. Despite of its relatively low price, such method has higher requirements for superficial modification of micro-fluidic materials in view of reducing noise signals produced by other non-target particles in food samples and interference to the detection signals.
Rolling circle amplification (RCA) is based on signal amplification technology on nucleic acid amplification, which has been used for detection of cells, proteins, and other small molecules. According to its working principles, it takes circular DNA as the template to convert deoxynucleotide (dNTPs) into single-stranded DNA products under the catalysis of DNA polymerase by using a primer complementary to both ends of the padlock probe (it can form a circular template). RCA products comprise hundreds or thousands of tandem repeated DNA fragments complementary to the template. As one of isothermal nucleic acids amplification techniques, RCA features in high reaction efficiency, moderate conditions and low operation cost of instruments as compared with Polymerase Chain Reaction (PCR). RCA is applied to signal amplification during detection of foodborne pathogens to effectively enhance detection signals. Objectively, detection sensitivity of such technology requires enhancement through improvement of identification and capture of targets.
With regard technologies on gene chips, aptamers are frequently used to identify and capture targets for detection. As compared with antibodies, aptamers as obtained through screening are available for in-vitro synthesis in large quantity, which features in excellent repeatability, high stability, easy storage and low cost. Activities of aptamers are not to be affected when used in combination with labelled fluorescence. Furthermore, the aptamers have extensive targets, including pesticides, tissues, cells, viruses, proteins, toxins, vitamins, allergens and so on. They are normally applied to aptamers of micro-fluidic chips, which are connected to the detection area in a single layer. However, such aptamers are unfavorable for contact with and identification of such large targets as bacteria cells due to their limited volume. Furthermore, their capture efficiency is also low.